Human exposure to environmental toxicants from contaminated food, soil, air, and water are an important environmental health problem worldwide. Such exposures have been linked to a number of human disorders including neurological and developmental defects and cancer. Epidemiological studies suggest that toxic heavy metals can induce nuclear and/or mitochondrial DNA damage resulting in oxidative stress that may lead to a constellation of deleterious biological events with adverse effects to organs such as kidney, lung, liver, intestine and brain. In order to combat such continuous insults, cells have evolved organelle specific damage tolerance pathways to execute an appropriate stress response mediated by mitochondrial and nuclear signaling networks. Characterizing such pathways and understanding how cells detect and respond to environmental stressors is not only biologically important but may also serve as the backbone for identifying early biomarkers of exposures and targets for clinical interventions aimed at prevention and treatment of human disease. However, the molecular components of mitochondrial and nuclear (retrograde and anterograde) signaling networks and the specific stress-response pathways that determine the differential outcomes (adaptive vs pathologic) are understudied and far from understood. A major limitation in understanding mitochondrial retrograde and nuclear anterograde signaling and stress response and how environmental toxins promote or exacerbate it, is the absence of suitable models in which to generate and monitor organelle specific, chemically identical, oxidative stress, which sensitizes both the nucleus and the mitochondrion with comparable selective reactivity. The major goal of this R21 proposal is to develop novel in vitro models of mitochondria and nuclear ROS signaling/damage pathways, by targeted delivery of ROS generating compounds (thiazole orange [TO]) to either the nucleus or the mitochondria, and visible light to increase the levels of either mitochondrial or nuclear ROS. Such a setup would model environmental toxin exposures and allow mitochondria-to-nucleus and nucleus-to-mitochondria signaling pathway signatures to be identified systematically. Using these signatures, we will then determine if environmental arsenic exposure in vitro evokes a mitochondrial or nuclear ROS response as part of its toxic mechanism. The results of this study will greatly expand our knowledge of signaling pathways that respond to the presence of oxidative stress in nuclei or mitochondria, determine how the signals exert their effects, and will inform future studies aimed at monitoring, diagnosing and perhaps counteracting these.